Microparticles for controlled growth differentiation factor 6 delivery to direct adipose stem cell-based nucleus pulposus regeneration.

Tom Hodgkinson,Judith A Hoyland,Jasmine Z Stening,Lisa J White,Kevin M Shakesheff,Stephen M Richardson

doi:10.1002/term.2882

Abstract

Currently, there is no effective long‐term treatment for intervertebral disc (IVD) degeneration, making it an attractive candidate for regenerative therapies. Hydrogel delivery of adipose stem cells (ASCs) in combination with controlled release of bioactive molecules is a promising approach to halt IVD degeneration and promote regeneration. Growth differentiation factor 6 (GDF6) can induce ASC differentiation into anabolic nucleus pulposus (NP) cells and hence holds promise for IVD regeneration. Here, we optimised design of novel poly(DL‐lactic acid‐co‐glycolic acid) (PLGA)–polyethylene glycol–PLGA microparticles to control GDF6 delivery and investigated effect of released GDF6 on human ASCs differentiation to NP cells. Recombinant human (rh)GDF6 was loaded into microparticles and total protein and rhGDF6 release assessed. The effect of microparticle loading density on distribution and gel formation was investigated through scanning electron microscopy. ASC differentiation to NP cells was examined after 14 days in hydrogel culture by quantitative polymerase chain reaction, histological, and immunohistochemical staining in normoxic and IVD‐like hypoxic conditions. RhGDF6 microparticles were distributed throughout gels without disrupting gelation and controlled rhGDF6 release over 14 days. Released GDF6 significantly induced NP differentiation of ASCs, with expression comparable with or exceeding media supplemented rhGDF6. Microparticle‐delivered rhGDF6 also up‐regulated sulphated glycosaminoglycan and aggrecan secretion in comparison with controls. In hypoxia, microparticle‐delivered rhGDF6 continued to effectively induce NP gene expression and aggrecan production. This study demonstrates the effective encapsulation and controlled delivery of rhGDF6, which maintained its activity and induced ASC differentiation to NP cells and synthesis of an NP‐like matrix suggesting suitability of microparticles for controlled growth factor release in regenerative strategies for treatment of IVD degeneration.

Highlights

Cell‐mediated regenerative therapy, in combination with delivery of small bioactive molecules, is an attractive strategy for the treatment of intervertebral disc (IVD) degeneration, a leading cause of low back pain (Balagué, Mannion, Pellisé, & Cedraschi, 2012; Cheung et al, 2009)
IVD degeneration arises from cell‐mediated changes in the extracellular matrix (ECM) of the central portion of the disc, the nucleus pulposus (NP)
The aim of this study was to encapsulate recombinant human GDF6 (rhGDF6) in novel poly(DL‐lactic acid‐co‐glycolic acid) (PLGA)/PLGA–polyethylene glycol (PEG)–PLGA MPs to control its release over a defined timescale in vitro

Summary

Introduction

Cell‐mediated regenerative therapy, in combination with delivery of small bioactive molecules, is an attractive strategy for the treatment of intervertebral disc (IVD) degeneration, a leading cause of low back pain (Balagué, Mannion, Pellisé, & Cedraschi, 2012; Cheung et al, 2009). Progressive injury, including tearing and fissure formation, to the AF and NP from increased localised compressive stress creates a proinflammatory environment, inflaming surrounding tissues and further increasing the expression of catabolic enzymes and factors (Johnson, Schoepflin, Choi, Shapiro, & Risbud, 2015; Risbud & Shapiro, 2014). This process is concomitant with in‐growth of blood vessels and nociceptive nerve fibres into the IVD, facilitating immune cell infiltration and thought to increase pain (Freemont et al, 1997; Hughes, Freemont, Hukins, McGregor, & Roberts, 2012)

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